Autophagy repression by antigen and cytokines shapes mitochondrial, migration and effector machinery in CD8 T cells

nature immunology Article https://doi.org/10.1038/s41590-025-02090-1 Autophagy repression by antigen and cytokines shapes mitochondrial, migration and effector machinery in CD8 T cells Received: 10 June 2024 Accepted: 15 January 2025 Linda V. Sinclair 1,4 , Tom Youdale1,4, Laura Spinelli 1, Milica Gakovic1, Alistair J. Langlands 2, Shalini Pathak1, Andrew J. M. Howden 1, Ian G. Ganley 3 & Doreen A. Cantrell 1 Published online: 27 February 2025 Check for updates Autophagy shapes CD8 T cell fate; yet the timing, triggers and targets of this process are poorly defined. Herein, we show that naive CD8 T cells have high autophagic flux, and we identify an autophagy checkpoint whereby antigen receptor engagement and inflammatory cytokines acutely repress autophagy by regulating amino acid transporter expression and intracellular amino acid delivery. Activated T cells with high levels of amino acid transporters have low autophagic flux in amino-acid-replete conditions but rapidly reinduce autophagy when amino acids are restricted. A census of proteins degraded and fueled by autophagy shows how autophagy shapes CD8 T cell proteomes. In cytotoxic T cells, dominant autophagy substrates include cytolytic effector molecules, and amino acid and glucose transporters. In naive T cells, mitophagy dominates and selective mitochondrial pruning supports the expression of molecules that coordinate T cell migration and survival. Autophagy thus differentially prunes naive and effector T cell proteomes and is dynamically repressed by antigen receptors and inflammatory cytokines to shape T cell differentiation. CD8 T lymphocytes shape transcriptional outputs by regulating protein synthesis and degradation. Naive and memory T cells have low levels of protein synthesis and T cell clonal expansion, and cytotoxic T cell (CTL) differentiation is driven by increases in protein synthesis that implement T cell transcriptional programs. Protein synthesis requires amino acids that are obtained from the environment by membrane amino acid transporters or supplied by autophagy, which degrades intracellular proteins to recycle amino acids. Autophagy is critical for naive CD8 T cell survival1–7 and memory cell formation7–10. Autophagy also restrains CTL function, and its loss promotes CD8 T cell anti-tumor immunity11,12. Given the importance of autophagy for CD8 T cells, it is essential to understand how this process is controlled and what proteins are degraded and fueled by autophagy in different CD8 T cell populations. Initial studies proposed that immune activation switches on autophagy1,2,13–15. These conclusions were based on measuring autophagosome levels or quantitating expression of autophagy markers such as microtubule-associated protein 1 light chain 3 beta (MAP1LC3b)16–20. By contrast, experiments with dynamic autophagy flux reporters observed low autophagy in proliferating T cells and high autophagy in memory T cells7. To resolve these discrepancies, the present study uses mass spectrometry and a dynamic autophagy reporter in tandem to comprehensively quantify autophagy machinery and autophagy flux as T cells differentiate. Our findings reveal that autophagy flux is high in naive T cells but rapidly repressed by antigen receptor engagement. Autophagy machinery expression is high in effector cells but autophagy flux is dynamic and repressed by pro-inflammatory cytokines but not by cytokines that promote the formation of memory T cells. The control of amino acid transporter Division of Cell Signalling and Immunology, School of Life Sciences, University of Dundee, Dundee, UK. 2National Phenotypic Screening Centre, School of Life Sciences, University of Dundee, Dundee, UK. 3MRC PPU, School of Life Sciences, University of Dundee, Dundee, UK. 4These authors contributed equally: Linda V. Sinclair, Tom Youdale. e-mail: ; 1 Nature Immunology | Volume 26 | March 2025 | 429–443 429 Article expression and intracellular amino acid delivery by antigen receptors and cytokines is identified as a critical autophagy checkpoint for T cells. A proteomic census also maps autophagy substrates and proteins fueled by autophagy in naive and effector CD8 T cells, which explains why precise regulation of autophagy allows CD8 T cells to initiate and curtail effector function. Results Immune-activated T cells accumulate autophagy machinery but repress autophagy flux To assess whether immune activation changes the abundance of autophagy machinery, we examined mass spectrometry data that quantitatively analyzed T cell proteomes21,22. These data show that the ATG8-family protein MAP1LC3b is not detected in naive T cells but is abundant in antigen-activated T cells and CTLs (Fig. 1a). CD8 T cells also increase the abundance of ATG8 proteins GABARAP and GABARAPL2 as they differentiate into CTLs (Extended Data Fig. 1a). The abundance of other core autophagy components also increases as CD8 T cells differentiate (Fig. 1b,c, Extended Data Fig. 1a and Source Data Fig. 1). Sequestosome-1 (p62/SQSTM1), an autophagy adaptor that targets proteins for degradation, accumulates in immune-activated cells (Fig. 1d), as do cargo adaptors for mitochondrial autophagy (mitophagy) and endoplasmic reticulum autophagy (ER-phagy) (Fig. 1c and Extended Data Fig. 1b). Autophagy protein accumulation occurs rapidly, within 3–6 h of antigen exposure (Fig. 1e), and does not correlate with an increased abundance of corresponding mRNA (Fig. 1a,d and Extended Data Fig. 1a,b). Cells with high autophagic flux constantly degrade autophagy machinery; autophagy proteins would be in low abundance when autophagy rates are high but would accumulate when autophagy is repressed. A low abundance of MAP1LC3b and GABARAPs in naive T cells and their accumulation in activated CD8 T cells could thus reflect that naive T cells exhibit high autophagy but repress autophagy as they respond to immune activation. To explore this hypothesis, we used an autophagy flux reporter that incorporates the normalization of autolysosome levels to accurately measure autophagy flux in primary tissues23,24. In this model, an mCherry–GFP–Map1lc3b (mCherry– GFP–LC3b) fusion protein is expressed ubiquitously from the ROSA26 locus. When autophagy initiates, tandem-tagged LC3b is recruited into autolysosomes, where low luminal pH quenches GFP fluorescence while mCherry fluorescence remains stable. Increased autophagic flux is visualized and quantified by measuring GFP fluorescence quenching normalized to mCherry fluorescence. Flow cytometry shows that cells not undergoing autophagy have linear GFP vs mCherry fluorescence profiles whereas cells undergoing autophagy have quenched (lower) GFP vs mCherry signals (Fig. 1f). Fig. 1 | Immune-activated T cells repress autophagy flux. a–d, Data are from naive, 24 h antigen-activated P14-CD8 T cells (TCR) and IL-2-maintained CTLs. a, MAP1LC3B protein copies per cell and mRNA (fragments per kilobase million (FPKM)). b, Summed copies of core au (...truncated)